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Minimum inhibitory concentration,Antibiotic Sensitivity Testing

Minimum inhibitory concentration,Antibiotic Sensitivity Testing

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    Minimum inhibitory concentration Minimum inhibitory concentration Presentation Transcript

    • Dr.T.V.Rao MD
      Antibiotic Sensitivity testing Minimum inhibitory concentrationskill based learning
      Dr.T.V.Rao MD
      1
    • The responsibility of the microbiology laboratory includes not only microbial detection and isolation but also the determination of microbial susceptibility to antimicrobial agents. Many bacteria, in particular, have unpredictable susceptibilities to antimicrobial agents, and their susceptibilities can be measured in vitro to help guide the selection of the most appropriate antimicrobial agent.
      Why antibiotic susceptibility testing
      Dr.T.V.Rao MD
      2
    • What is susceptibility to Antibiotics
      The term susceptible means that the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose of the antimicrobial agent and implies that an infection caused by this microorganism may be appropriately treated with the antimicrobial agent. The term resistant indicates that the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agent.
      Dr.T.V.Rao MD
      3
    • Antimicrobial resistance results from ….
      Results from misuse, overuse, under/ inadequate use of
      antimicrobials
      Costs money, lives and undermines effectiveness of health delivery programs
      Threat to global stability and national security
      WHO Global Strategy for Containment of Antimicrobial
      Resistance:
      Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganisms
      Dr.T.V.Rao MD
      4
    • Antibiotic resistant infections
    • Antimicrobial susceptibility tests
      Minimum inhibitory concentration [MIC]
      The smallest concentration of antibiotic that inhibits the growth of organism
      Liquid media (dilution) allows MIC estimation
      Solid media (diffusion)
      Disk diffusion (Kirby-Bauer)
      E-tests
      Allows MIC estimation
      Beta lactamase production: quick screening method
      Dr.T.V.Rao MD
      6
    • Dr.T.V.Rao MD
      7
    • Susceptibility testing: The big gaps
      MICs help, but hard to standardize
      Correlations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lacking
      Understanding this helps a lot when trying to correlate outcome with MIC
      - Some patients get better despite MICs
      - Some patients just don’t get better despite MICs
      No rule when it correlates/not correlates
      Dr.T.V.Rao MD
      8
    • The tube dilution test is standard method
      The tube dilutiontest is the standard method for determining levels of microbial resistance to an antimicrobial agent Serial dilutions of the test agent are made in a liquid microbial growth medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the minimal / minimum inhibitory concentration (MIC). At this dilution the test agent is bacteriostatic.
      Dr.T.V.Rao MD
      9
    • The MIC or minimum inhibitory concentration test determines antimicrobial activity of a material against a specific bacteria.
      The most commonly employed methods are the tube dilution method and agar dilution methods. Test products that are not clear or precipitate the growth media are tested by agar dilution methods which is about same as tube dilution method except dilutions are plated on agar
      minimum inhibitory concentrationtest determines
      Dr.T.V.Rao MD
      10
    • The Minimum Inhibitory Concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure only provides interval censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors
      Minimum Inhibitory Concentration (MIC)
      Dr.T.V.Rao MD
      11
    • The minimal bactericidal concentration (MBC) or the minimum lethal concentration (MLC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by sub culturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media.
      The minimal bactericidal concentration
      Dr.T.V.Rao MD
      12
    • A minimum bactericidal concentration test
      Figure 10.12
      Dr.T.V.Rao MD
      13
    • Minimum inhibitory concentration (MIC)Practice the exercise for skills
      1.    Grow cultures up overnight.2.    The following day, inoculate a fresh culture with a 1:10-20 dilution and grow up to OD600 0.4. 3.    Dilute to OD600 0.0005 (1:800) in THB (or relevant media).4.    Dilute this 1:200 and drop about 25µl on THA (or relevant agar plates) to confirm equal starting inoculums.5.    Put 50µl of the 0.0005 cultures in a 96 well plate in triplicates.6.    Add 50µl per well of the substance you want to test (ie. antimicrobial peptide, antibiotic) to each well, keeping in mind the final concentration will be half the original concentration you are adding. It is good to test 1:2 dilutions (in THB, or relevant media) of this substance.7.    Incubate overnight and check by eye or OD600 24 hours later to determine what concentration inhibited the growth of the different strains of bacteria.
      Dr.T.V.Rao MD
      14
    • Dilution in liquid broth
      MIC
      Tubes containing increasing antibiotic concentrations
      Incubation during 18 hr at 37°C
      Tedious
      Bacterial growth
      Inhibition
      0 (Control) 0,25 0,50 1 2 4 8 mg/l
      Dr.T.V.Rao MD
      15
    • Dr.T.V.Rao MD
      16
    • Dr.T.V.Rao MD
      17
    • Standard strains for quality assurance
      Precision and accuracy ensured through control strains
      Known susceptibility to antimicrobial agents
      Standard strains include
      Staphylococcus aureus ATCC 25923
      Escherichia coli ATCC 25922
      Pseudomonas aeruginosa ATCC 27853
      Dr.T.V.Rao MD
      18
    • Use standardized reference
      National Committee for Clinical Laboratory Standards (USA)
      Other norms
      Canadian
      Chinese
      National
      Do not confuse the different tables
      Choose one for everything
      Different standardsCLSI Standards followed all over the world
      Dr.T.V.Rao MD
      19
    • Culture media: Muller-Hinton
      Reagents: disks
      Size of the inoculums
      Incubation condition
      Control with reference strains
      Reading inhibition diameters (accurate measurement)
      Knowledge of staff
      Critical points in quality assurance- Organise your Laboratory with
      Dr.T.V.Rao MD
      20
    • Principles of Minimum inhibitory concentration
      In minimum inhibitory concentration dilute on a log2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a micro plate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration (MIC). The MIC and the zone diameter of inhibition are inversely correlated . In other words, the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC and the larger the zone of inhibition. Conversely, the more resistant the microorganism, the higher the MIC and the smaller the zone of inhibition.
      Dr.T.V.Rao MD
      21
    • Minimum Inhibitory Concentration (MIC) :
      Principle:
      • The tube dilution test is the standard method for determining levels of resistance to an antibiotic.
      • Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time.
      • The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).
      Dr.T.V.Rao MD
      22
      • Different concentrations of Gentamycin in Nutrient broth:
      Conc. in mcg/ml
      0.1 0.2 0.4 0.8 1.6 3.1
      Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml
      Dr.T.V.Rao MD
      23
      • Different concentrations of Tetracycline in Nutrient broth:
      Conc. in mcg/ml
      0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5
      Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml
      Dr.T.V.Rao MD
      24
    • Minimum inhibitory concentration test
      Figure 10.10
      Dr.T.V.Rao MD
      25
    • Interpretation
      The main concept is the “clinical categorisation"
      Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints
      c and C are the minor and major breakpoints
      Dr.T.V.Rao MD
      26
    • Understanding breakpoints
      Words of laboratory specialists
      It is not possible to work alone
      Breakpoints are the expression of a consensus among the scientific community at a given time in a country
      Breakpoints are determined using two approaches
      Pharmacological concept
      Epidemiological concept
      Dr.T.V.Rao MD
      27
    • Inherited resistance mechanism
      Wild type
      c
      C
      MIC
      The epidemiological concept for breakpoints
      Dr.T.V.Rao MD
      28
    • The pharmacological concept for breakpoints
      The concentration range tested for a drug and the
      interpretative criteria for various categories are based on
      extensive studies that correlate with
      Serum achievable levels for each antimicrobial agent
      Particular resistance mechanisms
      Successful therapeutic outcome
      In practice situations the entire range may not be used for
      decision making and therefore the concept of breakpoint
      concentration
      Dr.T.V.Rao MD
      29
    • From breakpoints to interpretation
      Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problem
      Defining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring
      Dr.T.V.Rao MD
      30
    • Sometime the agent can still be used
      Higher doses required to ensure efficacy
      Agent may be efficacious if concentrated in vivo in an infected body fluid (e.g., urine)
      Sometimes there is uncertainty
      Intermediate resistance may represent a “buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant
      Interpreting intermediate resistance
      Dr.T.V.Rao MD
      31
    • Common interpretation problems
      Results depends on the technique used
      Many factors influence results
      Lack of standardization of the inoculums
      Thickness and quality of the culture media
      Quality and conservation of the disks
      Quality control with standardized strains
      Condition and duration of incubation
      Dr.T.V.Rao MD
      32
    • E-test is based on arraying a concentration gradient of each antibiotic on a polymer strip. Concentration values are marked on the other side of the strip so that one can easily locate corresponding concentrations. E-strips, also known as “epsilometers”, are commercially prepared by micro dispersing robotic machines that can deliver Nano liter volumes of antibiotic concentration along the strip.
      What is e-test
      Dr.T.V.Rao MD
      33
    • E-test
      Plastic strips with a predefined gradient of
      One antibiotic
      One antifungal
      Only one manufacturer
      One strip per antibiotic
      Wide range of antibiotics
      Easy to use
      Storage at -20°C
      Short shelf life, expensive
    • An E-test combines aspects of Kirby-Bauer and MIC tests
      Figure 10.11
      Dr.T.V.Rao MD
      35
    • Reading E-tests
      Ciprofloxacin for Yersinia pestis
      Resistant > 4 ug/ml
      Intermediate 1-4 ug/ml
      Susceptible < 1
      Upper reading
      Dr.T.V.Rao MD
      36
    • Problems with E-test reading
      Common interpretation problems
      Dr.T.V.Rao MD
      37
    • The Liofilchem MIC Test Strip is a quantitative assay for determining the Minimum Inhibitory Concentration (MIC) of antimicrobial agents against microorganisms to indicate appropriate patient treatment and for identifying resistance patterns
      New Liofilchem MIC Test Strips
      Dr.T.V.Rao MD
      38
    • When the Liofilchem MIC Test Strip is applied onto an inoculated agar surface, the preformed exponential gradient of antimicrobial agent is transferred into the agar matrix. After 18 hours incubation or longer, a symmetrical inhibition ellipse centered along the strip is formed. The MIC is read directly from the scale in terms of µg/mL, at the point where the edge of the inhibition ellipse intersects with the MIC Test Strip.
      New Liofilchem MIC Test Strips
      Dr.T.V.Rao MD
      39
    • Oxoid has l improved M.I.C.Evaluator™ (M.I.C.E.™) strips, a product range for the accurate determination of minimum inhibitory concentration (MIC) values. The new distinctive gradient format of M.I.C.E. strips provides an excellent contrast with agar and the increased font size makes reading easier.
      M.I.C.Evaluators (M.I.C.E.) Simple, Convenient Method for Accurate MIC Values
      Dr.T.V.Rao MD
      40
    • Role of NCDC ( INDIA )in containment of Antimicrobial resistance (AMR)
      Antimicrobial resistance in pathogens causing important communicable diseases has become a matter of great public health concern globally including our country. Resistance has emerged even to newer, more potent antimicrobial agents like carbapenems. The factors responsible for this are widespread use and availability of practically all the antimicrobials across the counter meant for human, animal and industrial consumption. There are definite policies /guidelines for appropriate  use of  antimicrobials at national level in specific national health programme being run in the country
      Dr.T.V.Rao MD
      41
    • Dr.T.V.Rao MD
      42
      Implementation of WHONET CAN HELP TO MONITOR RESISTANCE
      Legacy computer systems, quality improvement teams, and strategies for optimizing antibiotic use have the potential to stabilize resistance and reduce costs by encouraging heterogeneous prescribing patterns and use of local susceptibility patterns to inform empiric treatment.
    • Antibiotic Resistance a concern to humanity ???
      Dr.T.V.Rao MD
      43
    • Dr.T.V.Rao MD
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      Follow me for Articles of Interest on Microbiology ..
    • Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the Developing World
      Email
      doctortvrao@gmail.com
      Dr.T.V.Rao MD
      45